One year experience of Achromobacter bacteremia at a tertiary care hospital in Northern India

Introduction. Achromobacter is a Gram-negative, motile, obligate aerobic and non-fermentative bacterium. It is an emerging pathogen in the hospital environment as it is frequently found in various solutions. Hypothesis/Gap Statement. Information about the incidence and risk factors of Achromobacter bacteremia from India is limited. Aim. We conducted this study to identify the risk factors and underlying conditions predisposing to bacteremia by Achromobacter spp. and analyse the antibiotic resistance pattern of the isolates. Methodology. We performed a retrospective observational study where automated blood cultures positive for Achromobacter spp. were assessed for clinical characteristics and antibiotic susceptibility patterns from January 2022 to December 2022 in the microbiology laboratory of a tertiary care centre in Northern India. Results. A total of 14 cases (14/2435, 0.57 %) of Achromobacter spp. were identified from bloodstream infections in one year. The mean age of the patients was 37.59±23.17 years with a male predominance (8/14, 57.1 %). All patients were managed on intravenous antibiotics and intravenous access as peripheral line catheters and only 5(5/14, 35.7 %) patients were managed on central line catheters. The isolates were found highly susceptible to ticarcillin-clavulanic acid (14/14, 100.0 %) followed by fluoroquinolones (12/14, 85.72 %) and trimethoprim-sulphamethoxazole (12/14, 85.72 %). Only 57.14 % (8/14, 57.14 %) of the patients were susceptible to piperacillin-tazobactam. The all-cause 40 day mortality was observed in 35.7 % (5/14, 35.7 %) with two deaths that were directly attributable to sepsis. Conclusion. This study provides insight into the incidence of Achromobacter bacteremia at our centre and the necessary antibiotic therapy to combat it.


INTRODUCTION
Achromobacter is a Gram-negative, motile, obligate aerobic and non-fermentative bacteria.It is catalase and oxidase positive, citrate positive, and indole and urease negative [1].Achromobacter species has various subspecies, among which the two most important clinically isolated species are xylosoxidans and denitrificans [2].Achromobacter xylosoxidans was first described by Yabuuchi and Ohyama et al. from an ear pus sample obtained from a patient suffering from chronic ear infections [3].
Achromobacter spp. is often misidentified by other non-lactose fermenting, Gram-negative bacteria, mostly Pseudomonas spp., and with rare pathogens (i.e.Pandoraea spp.and Ralstonia spp.) with the use of conventional methods due to similar biochemical reactions [4][5][6].Achromobacter is a multidrug-resistant (MDR) opportunistic pathogen [7].It is known to be an emerging pathogen in the hospital environment as it is frequently found as a contaminant in various solutions used in hospitals including water from nebulizers, dialysis water, incubators, extracorporeal circulation system and also in poorly preserved antiseptic and disinfectant solutions due to resistance to these agents [8,9].Achromobacter spp.most predominantly Achromobacter xylosoxidans, is recovered as a chronic respiratory pathogen from patients with cystic fibrosis (CF) attributable to poor patient outcome [10][11][12].Many authors have reported a high prevalence of Achromobacter in hospital settings as a result of cross-contamination [12,13].Achromobacter spp. is an MDR pathogen with intrinsic resistance to various antibiotics including aminoglycosides, most cephalosporins, aztreonam by virtue of multidrug efflux pumps, and chromosomal OXA-114-like β-lactamases.It also illustrates increased drug resistance to carbapenems by virtue of AmpC type β-lactamases, and metallo-β-lactamases (MBLs) [14].The MDR nature of bacteria and lack of data on appropriate therapy makes treating these bacteria difficult in hospital settings.Our study intended to recognize the risk factors and underlying comorbidities predisposing to bacteremia by Achromobacter spp.and furthermore analyse the pattern of antibiotic resistance among these isolates.

Study design and study population
We performed a retrospective observational study where automated blood cultures positive for Achromobacter spp.were assessed for clinical characteristics and antibiotic susceptibility patterns from January 2022 to December 2022 in the microbiology laboratory of a university hospital in Northern India.The study protocol was approved by the Institutional Ethics Committee (2021-52-EMP-EXP Dated 13/04/21).Informed consent was waived off given our study is retrospective.

Data collection
A set of blood culture bottles were received in the Department of Microbiology and sent to the Bacteriology section for standard microbiological processing like culture using the BACTEC blood culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) followed by microscopy for bacterial isolation.Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and (VITEK MS, bioMérieux, USA) were the diagnostic modalities used for confirmed identification of Achromobacter spp. in this study.

Inclusion criteria
Only a true pathogen was incorporated in our study.A true pathogen was defined based on the time of positivity of blood culture and clinical and laboratory indexes.Using the National Healthcare Safety Network definitions, the blood cultures that flagged positive when sent to the laboratory within 48 h of admission were labelled as community-acquired BSI and the blood cultures that flagged positive when sent to the laboratory after 48 h of admission were labelled as nosocomial in origin [15].

Exclusion criteria
Isolates collected only on a single positive culture without any significant clinical parameters were considered a contaminant and excluded from the study.

Treatment administered
We considered the use of appropriate antibiotics even if one drug from the panel was used in the treatment of the patient.

Statistical analysis
Categorical variables were described using percentages and a Chi-square test was applied.Univariate analysis of the risk factors in all patients included in our study was assessed using Kaplan-Meier survival analysis.All statistical analyses were performed using SPSS statistical software (IBM SPSS version 20, Armonk, N.Y.).
We also investigated the source of bacteremia in the patients included in our study cohort.Most patients included in the study were suffering from nosocomially acquired bacteremia (10/14, 71.43 %) whereas only a fraction of patients were suffering from community acquired bacteremia (4/14, 28.57%).All patients (14/14, 100.0 %) in this study were managed on intravenous antibiotics and thus intravenous access in the form of peripheral line catheters, and only 5 (5/14, 35.7 %) patients were managed using central line catheters.Other than catheters, pneumonia due to Achromobacter spp. was observed in two patients that were managed on mechanical ventilation (Table 1).No polymicrobial bacteremia or mixed infections were observed in our patient cohort.Empirical antibiotics were also administered after giving samples for blood culture.A total of 12 (12/14, 85.71 %) patients were treated with a combination therapy including meropenem-cilastatin and teicoplanin to cover both the Gram-negative and Gram-positive bacteria.Carbapenems (9/14, 64.28 %) were the most commonly administered antibiotic followed by thirdgeneration cephalosporins (6/14, 42.86 %) and β-lactam/β-lactam inhibitors (6/14, 42.86 %).
The mortality observed in our study was 35.7 % (5/14, 35.7 %).We compared the demographic and clinical characteristics among the Achromobacter bacteremia patient who died and survived in Table 3.Among the underlying comorbidities, electrolyte imbalance and cerebrovascular disease in the form of stroke were statistically significant risk factors associated with death in our study cohort.Neutropenia (P value=0.009) was identified as the most significant predisposing factor responsible for increased death in Achromobacter bacteremia patients.Empirical administration of antibiotics was done in patients after blood culture samples were taken.Carbapenems administration (P value=0.038) was more significantly associated with patients that died.Procalcitonin levels (P value=0.05)were found to be significantly raised in patients who died.
The all-cause 40 day mortality was observed in 35.7 %(5/14, 35.7 %) with two deaths, which were directly attributable to sepsis.We applied the Kaplan-Meier survival analysis on various risk factors observed in Achromobacter bacteremia and found renal failure (P value=0.024)as a significant risk factor in patients who died due to Achromobacter bacteremia (Fig. 2).
Achromobacter xylosoxidans is an unusual pathogen in many large studies involving the analysis of Gram-negative bacteria isolated from bloodstream infections [17,18].Other species of Achromobacter and Alcaligenes are known as causative agents in less than 2 % of cases of bacteremia [19,20].Three (3/14, 21.43 %) cases of Achromobacter xylosoxidans and 11(11/14, 78.57%) cases of Achromobacter denitrificans (Fig. 1) were isolated from bloodstream infections in this study.The cause of rare cases of Achromobacter bacteremia could be attributed to the absence of these micro-organisms from the normal skin flora of patients with immunocompromised conditions that could get infected bacteria from their skin flora [21,22].
Studies by Igra-Siegman et al. [23] and Vartivarian et al. [22] reported urinary tract infections with Achromobacter spp.while we did not encounter any cases of urinary tract infections in our study cohort.Pneumonia caused by this micro-organism was attributable to high case fatality among the patients.We identified 2 (2/14, 14.28 %) cases of pneumonia attributable to secondary bacteremia with Achromobacter spp.both were intubated during their hospital stay and further succumbed to their infections as a case fatality rate of 67 % was reported in a study by Morrison et al. in patients suffering with Achromobacter pneumonia [24].
Intravenous catheters were present in all 14 patients and removal of peripheral intravenous catheters was found to be effective in 7 (7/9, 77.78 %) out of 9 patients on intravenous access from peripheral sites whereas 2 patients succumbed to their ailment.Five (5/14, 35.71 %) patients were managed on central line catheters and did not undergo removal of the catheter, and were managed on antibiotics with an outcome of 60 %(3/5, 60.0 %) mortality.Similarly, bacteremia was observed to recur and high mortality was noted in patients with a catheter in situ with only 2 (2/5, 40.0 %) patients that were treated only on antibiotic therapy.Procalcitonin levels (P value=0.05)were found to be significantly raised in patients who died, owing to the fact that procalcitonin is denoted as a marker of sepsis, which is recognized as a common cause of death in hospitalized patients [25].
Achromobacter spp. is known to be resistant to aminoglycosides, older cephalosporins and narrow-spectrum penicillins and susceptible to sulphonamides, third-generation cephalosporins, and broad-spectrum penicillins and variably susceptible to fluoroquinolones [26,27].The Achromobacter spp.isolated from the bloodstream of patients included in our study was 100 %(14/14, 100.0 %) susceptible to ticarcillin-clavulanic acid, 85.72 %(12/14, 85.72 %) susceptible to trimethoprim-sulphamethoxazole (cotrimoxazole) and ciprofloxacin.Most Achromobacter spp.(8/14, 57.14 %) isolated from blood culture samples in our study showed in vitro susceptibility to piperacillin-tazobactam, which corroborates with studies by Turel et al. [28] and Nakamoto et al. [29].We also reported a case of   prosthetic valve Achromobacter endocarditis susceptible to piperacillin-tazobactam and trimethoprim-sulphamethoxazole, which corroborates with the susceptibility pattern of a previous reported case from our institute [30] and a case series by Liu et al. [31].We come across Achromobacter spp.isolates as highly (12/14, 85.72 %) susceptible to trimethoprim-sulphamethoxazole, which agrees with studies by D'Amato et al. [32] and Olson et al. [33].In contrast to the increasing resistance to fluoroquinolones in recent studies by Isler et al., all (14/14, 100 %) isolates in our study were susceptible to ciprofloxacin [34].Carbapenems administration (P value=0.038) was more significantly associated with patients that died, which could be attributed to various efflux pump mechanisms and metalloβ-lactamases leading to extrusion or inactivation of the drugs rendering it ineffective [35].
The overall mortality observed in our study was 35.71 % (5/14, 335.71 %).We performed Kaplan-Meier survival analysis on the patients with Achromobacter bacteremia in our study cohort and identified the absence of renal failure in the patient cohort as a statistically significant cause of increased survival in Achromobacter bacteremia patients.A similar outcome with a mortality rate of 33.33 %(5/15, 33.33 %) was observed in a study by Liu et al. [31].
The limitations of our study firstly include its retrospective nature, which can lead to the introduction of information bias into the study.Secondly, the data represented in this study only signifies the prevalence of Achromobacter bacteremia at a single hospital and does not represent the incidence of Achromobacter bacteremia in the geographical region.

CONCLUSION
This study provides insight to clinicians into the incidence of Achromobacter bacteremia at our centre and the necessary antibiotic therapy to combat it.This isolate is rarely identified from bloodstream infections in immunosuppressed patients and renal failure was identified as a prominent risk factor leading to increased 40 day mortality.
Comments: I see the revised version of this manuscript is suitable for publication in Access Microbiology.

Please rate the manuscript for methodological rigour Good
Please rate the quality of the presentation and structure of the manuscript

Very good
To what extent are the conclusions supported by the data?Strongly support

Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No The incidence of Achromobacterbacteremia requires multicenter study, as the authors mentioned in the limitations section.
The incidence of Achromobacterbacteremia requires multicenter study as the incidence was found to be low in single centre studies.
We have found the incidence of Achromobacter sppat our centre and highlighted it in the revised manuscript.A Multicenter study is being planned further.
2 Also, the authors didn't mention the incidence in their center.
We have mentioned and highlighted the incidence of Achromobacterbacteremia in the revised manuscript.No polymicrobial bacteremia or mixed infections were observed in our patient cohort and this statement has been mentioned in the manuscript and also highlighted in the revised manuscript.

4
The authors didn't mention, did the cases admitted to the hospital with Achromobacter infection or acquired it during the hospital stay?
A statement has been added to the revised manuscript to denote the source of infection and identify whether most infections were acquired during hospital stay."Most patients included in the study were suffering from nosocomially acquired bacteremia (10/14, 71.43%) whereas only a fraction of patients were suffering from community acquired bacteremia (4/14, 28.57%)".

5
For the conflicts of interest section: Please acknowledge their is no conflicts of interest.
A conflict of interest section has been added to the manuscript and no conflict of interest was there in this study.

6
For the consent of publication section: All individuals have given consent to participate in the study.& the author mentioned in the methods section: Informed consent was waived off given our study is retrospective -please explain this fully For the consent of publication section All authors have given the consent to participate in the study and it has been corrected in the revised manuscript.Informed consent was waived off given our study is retrospective study-it means that the patients were included in this study from previous laboratory and electronic records of the institute so in their absence at the present time, their consent was waived off being a retrospective study. 7 Please update the references.All the references have been updated and adjusted according to the requirement of the journal.4

Revision by
Were all positive blood cultures (for the true pathogens) within the one year of study period considered for analysis?If not, which sampling techniques were used to come up with the 14 cases?
We have included positive blood cultures to find the incidence of Achromobacter sppBSI in this study but not included all the demographics of patents with other pathogens in the past one year of study.In this study we have specifically analyzed the demographic and clinical characteristics of only those patients who were suffering from Achromobacter sppbacteremia.No sample technique was used to come up with the 14 cases. 5 Is there specific age bracket that is more vulnerable to infection with Achromobacterbacteria?Kindly justify the age choice for your study population.
No, there is no specific age bracket that is more vulnerable to infection with Achromobacterbacteria.We recorded the age of patients in our study population based on isolation characteristics of Achromobacter sppfrom bloodstream infections.

6
This finding… "Carbapenems administration (p-value= 0.038) was more significantly associated with patients that died.Procalcitonin levels (p-value= 0.05) were found to be significantly raised in patients who died" needs to be explained under the discussion section.
The explanation and correlation with previous study has been added and highlighted in the discussion section of the revised manuscript.

Fig. 2 .
Fig. 2. KaplanMeier urvival analysis of 40 day survival of the Achromobacter bacteremia patients suffering from renal failure in comparison to patients not suffering from renal failure (N=14)

Table 1 .
Demographic and clinical characteristics of patients with Achromobacter bacteremia (N=14)

Table 2 .
Results of in vitro susceptibility testing of Achromobacter spp.isolates (n=14)

Table 3 .
Comparison of demographic and clinical characteristics among the Achromobacter bacteremia patient who died and survived (n=14)

response to reviewers to Version 1 Response to reviewers' comments Revision by Reviewer 1 Reviewer 1 Comments to Author Response to reviewer 1
there a potential financial or other conflict of interest between yourself and the author(s)?This is an open-access article report distributed under the terms of the Creative Commons License.The Authors.This is an open-access article report distributed under the terms of the Creative Commons License.
5  © 2023Brown H.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.The reviewers have highlighted minor concerns with the work presented.Please ensure that you address their comments.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.